Method for generating batches of allogeneic T-cells with averaged potency

ABSTRACT

The present invention relates to a method for generating batches of lymphocytes with averaged potency. In particular, the present invention relates to a method of pooling lymphocytes from different donors to avoid NK alloreactivity and anti-HLA immune response. Lymphocytes from each donor are inactivated for at least a gene encoding a TCR component, and are pooled together before be administrated to a subject in need thereof. Thus, this method allows generating batches of lymphocytes with averaged potency, particularly to treat cancer, viral infection or auto-immune disease. The present invention also relates to a batch of lymphocytes obtainable by this method. The batch of lymphocytes can be used to be administrated to one or several patients, being made available as an “off the shelf” therapeutic product, in particular to treat cancer, auto-immune disease or viral infection.

FIELD OF THE INVENTION

The present invention relates to a method for generating batches of lymphocytes with averaged potency. In particular, the present invention relates to a method of pooling lymphocytes from different donors to avoid NK alloreactivity and anti-HLA immune response. Lymphocytes from each donor are inactivated for at least a gene encoding a TCR component, and are pooled together before be administrated to a subject in need thereof. Thus, this method allows generating batches of lymphocytes with averaged potency, particularly to treat cancer, viral infection or auto-immune disease. The present invention also relates to a batch of lymphocytes obtainable by this method. The batch of lymphocytes can be used to be administrated to one or several patients, being made available as an “off the shelf” therapeutic product, in particular to treat cancer, auto-immune disease or viral infection.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.

The current protocols for treatment of patients using adoptive immunotherapy is based on autologous cell transfer. In this approach, T lymphocytes are recovered from patients, genetically modified or selected ex vivo, cultivated in vitro in order to amplify the number of cells if necessary and finally infused into the patient. Autologous therapies face substantial technical and logistic hurdles to practical application, their generation requires expensive dedicated facilities and expert personnel, they must be generated in a short time following a patient's diagnosis, and in many cases, pretreatment of the patient has resulted in degraded immune function, such that the patient's lymphocytes may be poorly functional and present in very low numbers. Because of these hurdles, each patient's autologous cell preparation is effectively a new product, resulting in substantial variations in efficacy and safety.

Ideally, one would like to use a standardized therapy in which therapeutic cells could be pre-manufactured, characterized in detail, and available for immediate administration to patients. Such standardized therapy can be performed by using allogeneic cells obtained from individuals belonging to the same species but which are genetically dissimilar.

However, the use of allogeneic cells presently has many drawbacks. Endogenous TCR specificities of allogeneic cells recognize the host tissue as foreign, resulting in graft versus host disease (GvHD), which can lead to serious tissue damage and death. T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. As for immunoglobulin molecules, the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of GVHD. In order to effectively use allogeneic cells, the inactivation of TCRalpha or TCRbeta can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD.

On the other hand, host allogeneic cells can be rapidly rejected by the host immune system, a process termed host versus graft rejection (HvG) and this substantially limits the efficacy of the transferred cells. Allograft rejection is dependent upon recipient T lymphocytes responding to highly polymorphic cell surface molecules encoded by the MHC genes (the HLA system in humans). Although, the HLA immune response can be avoided by suppressing patient's immune system, the potency of allogeneic T cell engraftment from a single donor is influenced by the previous immunologic experience of the patient and the efficacy of engraftment is variable and not predictable. Indeed, patients who have been pregnant or who have had a blood transfusion may have already developed immunological memory and circulating antibodies to non-self HLA molecules and will, thus, be “sensitized” against certain HLA molecules. Thus, in general case of organ transplantation, graft rejection is triggered by anti-HLA antibodies. The graft rejection may be avoided by matching donor and recipient MHC (HLA) molecules. In practical terms, this has been managed 1) by performing a cell typing to determine the HLA genotype of donors and recipient prior engraftment, in order to select the closest HLA match and 2) by detecting circulating antibodies in the recipient's serum against HLA molecule on the donor lymphocytes. To avoid the typing of recipient T cells and the detection of circulating antibodies in the recipient's serum against HLA molecule on the donor's lymphocytes, the inventors propose for the first time to develop allogeneic immunotherapy product by generating batches of T cells originating from different donors.

The present method should allow obtaining a clinical response with averaged potency. Indeed, to avoid compromise of donor lymphocytes in patients with anti-HLA antibodies, lymphocytes selected to express varied HLA types are engrafted, thus preventing a high proportion of donor's cell to be subject to the same anti-HLA antibody. In addition, Natural Killer (NK) cells recognize HLA class I molecules via surface receptors killer immunoglobulin-like receptor (KIR) delivering signals that inhibit NK cell function. These receptors prevent NK cell-mediated attack against normal (i.e. HLAclass I⁺) autologous cells. Cells in which expression of HLA class I is different from autologous HLA become susceptible to NK-mediated killing.

The proper pooling of lymphocytes originating from different donors expressing varied HLA alleles, according to the invention, i.e. by taking into account variability among donors and patients, ensures that at least one fraction of the lymphocytes express appropriate MHC class I alleles that engage KIR and thus avoid a significant impact of NK alloreactivity on lymphocytes engraftment. In summary, the batch of lymphocytes originating from different donors minimizes impacts of existing anti-HLA immunoreactivity in patients, and reduces NK alloreactivity to allow clinical response with averaged potency.

SUMMARY OF THE INVENTION

The present invention relates to a new therapeutic strategy based on the use of batches of T-cells with averaged potency originating from different donors intended for administration as an “off the shelf” allogeneic treatment. Lymphocytes of these batches are characterized in that they do not express TCR at the surface of the cell to avoid GVHD and are originated from different donors to minimize effect of anti-HLA immunoreactivity in patients and reduces NK alloreactivity. This method more particularly comprises the step of: obtaining lymphocytes samples from individual donors; inactivating at least one gene encoding a TCR component into the cells of each individual sample; purifying the TCR negative cells from the sample; and pooling at least two samples to obtain a batch of T cells with averaged potency. The batches of T-cells obtained by this method are particularly suitable to treat tumor or viral infection with averaged potency.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Method for Generating a Batch of Lymphocytes with Averaged Potency

In a general aspect, the present invention relates to a method for generating at least one batch of inactivated TCR lymphocytes originating from different donors to treat cancer or infections with averaged potency. In particular, said method comprises the step of: obtaining lymphocyte samples from individual donors; inactivating at least one gene encoding a TCR component in the cells of each individual sample; purifying the TCR negative cells from the sample; and pooling at least two samples to obtain a batch of T cells. The pooling of lymphocytes from different donors allows to obtain a batch suitable for an “off the shelf treatment” with averaged potency.

Indeed, T-cell potency originating from a single donor may be influenced by the previous immunologic experience of the patient. Patients who have been subject to a previous transplant, blood transfusions, or pregnancies may express anti-HLA antibodies, which could lead to depletion of cells expressing the corresponding HLA type. Inventor's approach to avoid the depletion of a high proportion of cells with such antibodies is to generate a batch of T cells from different donors selected to express varied HLA types. On the other hand, the T-cell potency from a single donor may also be influenced by NK alloreactivity. Natural Killer (NK) cells recognize HLA class I molecules via KIR delivering signals that inhibit NK cell function. T-cells originating from a single donor in which expression of HLA class I is inappropriate become susceptible to NK-mediated killing. The generation and the use of a batch of T cells originating from different donors, preferably selected to express varied HLA types increase the probability to have a population of cells which express appropriate HLA class I molecule recognized by the KIR of NK cells, and thus inhibit the attack of the NK cells. Thus, the pooling strategy ideally focuses on maintaining sufficient HLA diversity to avoid NK alloreactivity and anti-HLA antibodies depletion of the major population of transplanted cells.

According to statistical analysis performed by the inventors, it has been established that, depending on the biological parameters considered as more important in the sought therapy and the chosen prioritization of those parameters, the number of donors could fall into different ranges. For instance, in situations where a minimal number of donors is sought for limiting the risk of infectious diseases, while providing sufficient diversity for a high probability of engraftment, sufficient HLA diversity can be obtained by pooling lymphocytes originating from at least three donors, preferably between 3 and 50 donors, more preferably between 3 and 30, even more preferably between 3 and 10 donors. Sufficient HLA diversity can be thus obtained by pooling lymphocytes originating from at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, up to 50 donors.

By contrast, in situations where it is sought for example that a given biological parameter stays in the final batch within an interval of 2 standard deviations, preferably one standard deviation, while the variability between donors is high for this parameter, better it is to pool between 30 and 150 donors, preferably between 50 and 150 donors, more preferably between 50 and 100 donors, even more preferably between from 50, 60 or 70 and 100 donors.

The HLA system of cell surface molecules is encoded within the MHC, a large group of genes on chromosome 6 in humans. It includes three highly polymorphic class I α-chain genes which combine with the beta-2-microglobulin (β2 m) chain to form the classical class I molecules HLA-A, HLA-B, and HLA-C, as well as three pairs of polymorphic class II α- and β-chain genes that combine to form the HLA-DR, -DP, and -DQ class II molecules. In addition to these loci, class III genes encode various serum proteins of the immune system, including components of the complement system. The cellular distribution of HLA molecules is determined largely by their function. Peptides presented by class I HLA molecules are derived from cytosolic proteins, such as viral pathogens and their gene products. Thus, HLA class I molecules are expressed constitutively by most nucleated cells of the body (since they are susceptible to viral attack) and, in particularly high density, by cells of the immune system. Peptides presented by class II molecules are generated by breakdown of endosomal proteins; class II expression is thus much less widespread and largely restricted to cells that have the capability to process and present exogenous soluble and particulate antigen. Cells with these characteristics include B lymphocytes, dendritic cells (DCs), macrophages, and monocytes; class II molecules may be induced, in inflammatory conditions, on certain types of epithelial and endothelial cells (ECs).

Each individual expresses only a relatively small number of HLA alleles: one allele each of HLA-A, -B and -C classical class I molecules from each parent together with one allele each of the three principal class II molecules (HLA-DR, -DP, -DQ) from each parent, with little or no crossover between the chromosomes supplying these alleles. The number of permutations of particular peptide-binding sites covered by these few alleles means that most potentially dangerous antigens may be presented and recognized by T cell receptors (TCRs) (for review Bolton Eleanor, Bradley, transplantation immunology, chapter 3, essential immunology for surgeons).

In a particular embodiment, to avoid NK alloreactivity, the batch of T-cells needs to express appropriate class I HLA molecule. In a preferred embodiment to minimize NK aloreactivity, the donors are selected to express at least one Bw4 motif of HLA-B and/or HLA-A allele, one C1 and/or one C2 class alleles in HLA-C loci. In a more preferred embodiment, the donors are selected to express at least one Bw4 motif of HLA-B and/or HLA-A loci and one C1 and one C2 alleles in HLA-C loci.

The pooling strategy is made possible according to the invention more particularly by inactivating at least one gene encoding a TCR component. TCR is rendered not functional in the cells by inactivating TCR alpha gene and/or TCR beta gene(s). This TCR inactivation allows pooling cells from different donors and avoiding GvHD. By inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form. In a preferred embodiment of the invention, the genetic modification of the method relies on the genetic inactivation of a gene encoding TCR by expression, in provided cells to engineer, of one rare-cutting endonuclease such that said rare-cutting endonuclease specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused by the rare-cutting endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts. Said modification may be a substitution, deletion, or addition of at least one nucleotide. Cells in which a cleavage-induced mutagenesis event, i.e a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known method in the art. In a particular embodiment, the step of inactivating at least a gene encoding a component of the T-cell receptor (TCR) into the cells of each individual sample comprises introducing into the cell a rare-cutting endonuclease able to disrupt at least one gene encoding a component of the T-cell receptor (TCR). In a more particular embodiment, said cells of each individual sample are transformed with nucleic acid encoding a rare-cutting endonuclease capable of disrupting at least one gene encoding a component of the T-cell receptor (TCR), and said rare-cutting endonuclease is expressed into said cells.

Said rare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease, CRISPR/Cas9 nuclease or a TALE-nuclease. In a preferred embodiment, said rare-cutting endonuclease is a TALE-nuclease. By TALE-nuclease is intended a fusion protein consisting of a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain to cleave a nucleic acid target sequence. (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al. 2011; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011; Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander, Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al. 2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al. 2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012). In the present invention new TALE-nucleases have been designed for precisely targeting relevant genes for adoptive immunotherapy strategies.

Preferred TALE-nucleases according to the invention are those recognizing and cleaving the target sequence selected from the group consisting of: SEQ ID NO: 1 to 5 (TCRalpha), SEQ ID NO: 6 and 7 (TCRbeta). Said TALE-nucleases preferably comprise a polypeptide sequence selected from SEQ ID NO: 8 to SEQ ID NO: 13. For a better and safer efficiency of the method, the cells are made allogeneic by transfection of said cells with mRNA molecules encoding said endonuclease targeting the TCR gene.

In another embodiment, additional catalytic domain can be further introduced into the cell with said rare-cutting endonuclease to increase mutagenesis in order to enhance their capacity to inactivate targeted genes. In particular, said additional catalytic domain is a DNA end processing enzyme. Non limiting examples of DNA end-processing enzymes include 5-3′ exonucleases, 3-5′ exonucleases, 5-3′ alkaline exonucleases, 5′ flap endonucleases, helicases, hosphatase, hydrolases and template-independent DNA polymerases. Non limiting examples of such catalytic domain comprise of a protein domain or catalytically active derivate of the protein domain selected from the group consisting of hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST). In a preferred embodiment, said additional catalytic domain has a 3′-5′-exonuclease activity, and in a more preferred embodiment, said additional catalytic domain is TREX, more preferably TREX2 catalytic domain (WO2012/058458). In another preferred embodiment, said catalytic domain is encoded by a single chain TREX2 polypeptide. Said additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein according to the invention optionally by a peptide linker.

Endonucleolytic breaks are known to stimulate the rate of homologous recombination. Thus, in another embodiment, the genetic modification step of the method further comprises a step of introduction into cells an exogeneous nucleic acid comprising at least a sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogeneous nucleic acid. In particular embodiments, said exogenous nucleic acid comprises first and second portions which are homologous to region 5′ and 3′ of the target nucleic acid sequence, respectively. Said exogenous nucleic acid in these embodiments also comprises a third portion positioned between the first and the second portion which comprises no homology with the regions 5′ and 3′ of the target nucleic acid sequence. Following cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used within said donor matrix. In a particular embodiment, the homologous sequence can be from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared nucleic acid homologies are located in regions flanking upstream and downstream the site of the break and the nucleic acid sequence to be introduced should be located between the two arms.

Chimeric Antigen Receptors

Chimeric Antigen Receptors (CAR) are able to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties. Thus, in another particular embodiment, the method further comprises a step of introducing into said lymphocytes a Chimeric Antigen Receptor. Said Chimeric Antigen Receptor combines a binding domain against a component present on the target cell, for example an antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-target cellular immune activity. Generally, CAR consists of an extracellular single chain antibody (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (scFv:ζ) and have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity. One example of CAR used in the present invention is a CAR directing against CD19 antigen and can comprise as non limiting example the amino acid sequence: SEQ ID NO: 14 or 15.

Immune-checkpoint Genes

T cell-mediated immunity includes multiple sequential steps involving the clonal selection of antigen specific cells, their activation and proliferation in secondary lymphoid tissue, their trafficking to sites of antigen and inflammation, the execution of direct effector function and the provision of help (through cytokines and membrane ligands) for a multitude of effector immune cells. Each of these steps is regulated by counterbalancing stimulatory and inhibitory signal that fine-tune the response. It will be understood by those of ordinary skill in the art, that the term “immune checkpoints” means a group of molecules expressed by T cells. These molecules effectively serve as “brakes” to down-modulate or inhibit an immune response. Immune checkpoint molecules include, but are not limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM_173799), LAIR1 (also known as CD305, GenBank accession number: CR542051.1, (Meyaard, Adema et al. 1997)), SIGLEC10 (GeneBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 (Nicoll, Ni et al. 1999), SIGLEC9 (Zhang, Nicoll et al. 2000; Ikehara, Ikehara et al. 2004), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF (Quigley, Pereyra et al. 2010), GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 which directly inhibit immune cells. For example, CTLA-4 is a cell-surface protein expressed on certain CD4 and CD8 T cells; when engaged by its ligands (B7-1 and B7-2) on antigen presenting cells, T-cell activation and effector function are inhibited.

Thus, the present method previously described of generating a batch of T-cells, can advantageously comprise the step of further modifying T-cells by inactivating at least one protein involved in a immune check-point, in particular PD1 and/or CTLA-4.

Immunosuppressive Resistant T Cells

Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008). Thus, to prevent rejection of allogeneic cells, the host's immune system has to be usually suppressed to some extent. However, in the case of adoptive immunotherapy the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be also resistant to the immunosuppressive treatment. Thus, in particular embodiment, the method according to the present invention further comprises a step of modifying T-cells to make them resistant immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. In other words, an immunosuppressive agent is a role played by a compound which is exhibited by a capability to diminish the extent of an immune response. As non limiting example, an immunosuppressive agent can be a calcineurin inhibitor, a target of rapamycin, an interleukin-2 α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. Classical cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act through activation of T-cells or by inhibiting the activation of helper cells. The method according to the invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non limiting examples, targets for immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member. In particular embodiment, the genetic modification of the method relies on the expression, in provided cells to engineer, of one rare-cutting endonuclease such that said rare-cutting endonuclease specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. Said rare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease or a TALE-nuclease. Preferred TALE-nucleases according to the invention are those recognizing and cleaving the target sequence selected from the group consisting of: SEQ ID NO: 16 to 21 (GR), and SEQ ID NO: 34 to 39 (CD52). Said TALE-nucleases preferably comprise a polypeptide sequence selected from SEQ ID NO: 22 to SEQ ID NO: 33 and SEQ ID NO: 40 to SEQ ID NO: 41.

Chemotherapeutic Agent Resistant T-cells

Although outstanding progress has been made in the fields of cancer detection and tumor cell biology, the treatment of late-stage and metastatic cancer remains a major challenge. Cytotoxic chemotherapy agents remain among the most used and successfully employed anti-cancer treatments. However, they are not uniformly effective, and the introduction of these agents with novel therapies, such as immunotherapies, is problematic. Thus, to improve cancer therapy and selective engraftment of T-cells, drug resistance can be conferred to said cells to protect them from the toxic side effects of chemotherapy agent. Thus, in another particular embodiment, the method according to the present invention further comprises modifying said T-cell to confer drug resistance. As used herein, a cell which is “resistant or tolerant” to an agent means a cell which has been genetically modified so that the cell proliferates in the presence of an amount of an agent that inhibits or prevents proliferation of a cell without the modification.

In a particular embodiment, said drug resistance can be conferred to the T-cell by the expression of at least one drug resistant gene. Said drug resistant gene refers to a nucleic acid sequence that encodes “resistance” to an agent, such as a chemotherapeutic agent (e.g. methotrexate).

In another particular embodiment, said drug resistance can be conferred to the T-cell by the inactivation of a drug sensitive gene. One potential interested drug sensitive gene which can be inactivated to confer drug resistance to the T-cell is the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (Gen bank: M26434.1). In particular HPRT can be inactivated in engineered T-cells to confer resistance to a cytostatic metabolite, the 6-thioguanine (6TG) which is converted by HPRT to cytotoxic thioguanine nucleotide and which is currently used to treat patients with cancer, in particular leukemias (Hacke, Treger et al. 2013). In another embodiment, the inactivation of the CD3 normally expresses at the surface of the T-cell can confer resistance to anti-CD3 antibodies such as teplizumab.

The therapeutic efficiency can be significantly enhanced by genetically engineering multiple drug resistant allogeneic T-cells. Such a strategy can be particularly effective in treating tumors that respond to drug combinations that exhibit synergistic effects. Moreover multiple resistant engineered T-cells can expand and be selected using minimal dose of drug agents. Thus, the method according to the present invention can further comprise modifying T-cell to confer multiple drug resistance to said T-cell.

Suicide Genes

In another aspect, since engineered T-cells can expand and persist for years after administration, it is desirable to include a safety mechanism to allow selective deletion of administrated T-cells. Thus, in some embodiments, the method of the invention can comprises the transformation of said T-cells with a recombinant suicide gene. Said recombinant suicide gene is used to reduce the risk of direct toxicity and/or uncontrolled proliferation of said T-cells once administrated in a subject (Quintarelli, Vera et al. 2007; Tey, Dotti et al. 2007). Suicide genes enable selective deletion of transformed cells in vivo. In particular, the suicide gene has the ability to convert a non-toxic pro-drug into cytotoxic drug or to express the toxic gene expression product. In other words, “Suicide gene” is a nucleic acid coding for a product, wherein the product causes cell death by itself or in the presence of other compounds. A representative example of such a suicide gene is one which codes for thymidine kinase of herpes simplex virus. Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil. Suicide genes also include as non limiting examples caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID). As used herein “prodrug” means any compound useful in the methods of the present invention that can be converted to a toxic product. The prodrug is converted to a toxic product by the gene product of the suicide gene in the method of the present invention. A representative example of such a prodrug is ganciclovir which is converted in vivo to a toxic compound by HSV-thymidine kinase. The ganciclovir derivative subsequently is toxic to tumor cells. Other representative examples of prodrugs include acyclovir, FIAU [1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil], 6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine for cytosine deaminase.

Delivery Methods

The different methods described above involve introducing a protein of interest such as rare cutting endonuclease into a cell. As non-limiting example, said protein of interest can be introduced as transgenes preferably encoded by at least one plasmid vector. Polypeptides may be synthesized in situ in the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into cells are known in the art and including as non limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods. Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells. Said plasmid vector can comprise a selection marker which provides for identification and/or selection of cells which received said vector. Different transgenes can be included in one vector. Said vector can comprise a nucleic acid sequence encoding ribosomal skip sequence such as a sequence encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip” from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see Donnelly et al., J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)). By “codon” is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA. As non-limiting example, in the present invention, 2A peptides have been used to express into the cell the rare-cutting endonuclease and a DNA end-processing enzyme.

A more preferred embodiment of the invention, polynucleotides encoding polypeptides according to the present invention can be mRNA which is introduced directly into the cells, for example by electroporation. The inventors determined the optimal condition for mRNA electroporation in T-cell.

The inventor used the cytoPulse technology which allows, by the use of pulsed electric fields, to transiently permeabilize living cells for delivery of material into the cells. The technology, based on the use of PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, Mass. 01746, USA) electroporation waveforms grants the precise control of pulse duration, intensity as well as the interval between pulses (U.S. Pat. No. 6,010,613 and International PCT application WO2004083379). All these parameters can be modified in order to reach the best conditions for high transfection efficiency with minimal mortality. Basically, the first high electric field pulses allow pore formation, while subsequent lower electric field pulses allow to move the polynucleotide into the cell.

Purifying T Cells

By purified T cells is meant that the ratio of T cells:hematopoietic cells in a purified T cell composition is increased in comparison to the ratio of T cells:hematopoietic cells in peripheral blood. In particular, by purified CD8+ T cells is meant that the ratio of CD8⁺ T cells:CD8⁻ T cells in a purified CD8+ T cell composition is increased in comparison to the ratio of CD8⁺ T cells:CD8⁻ T cells in peripheral blood. By purified CD4+ T cells is meant that the ratio of CD4⁺ T cells:CD4⁻ T cells in a purified CD4+ T cell composition is increased in comparison to the ratio of CD4⁺ T cells:CD4⁻ T cells in peripheral blood. Preferably, the purified T cells (CD8+ or CD4+) comprise at least 75%, more preferably at least 90% and most preferably at least 95% or even at least 99% of all T cells present in the composition. Methods for purifying CD4+ or CD8+ T cells are known to those of skill in the art. The expression of surface markers facilitates identification and purification of these cells. These methods of identification and isolation include FACS, column chromatography, panning with magnetic beads, western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno fluorescent assays, and the like. For a review of immunological and immunoassay procedures in general, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology (7th ed.) and Paul supra. In a preferred embodiment, the T cells are purified by magnetic sorting.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. Generally, the T cells of the invention are expanded by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T-cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T-cell. As non limiting examples, T cell populations may be stimulated in vitro such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. For example, the agents providing each signal may be in solution or coupled to a surface. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell.

Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, −10, −2, 1L-15, TGFp, IL-21 and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% C02). T cells that have been exposed to varied stimulation times may exhibit different characteristics.

Batch of T Cells with Averaged Potency

The present invention also relates to a batch of T-cells with averaged potency obtainable by the method described above. By “averaged potency” it is meant that considering infusing different patients with a dose of cells fractioned from the same batch or from different batches according to the invention, these cells with provide an engraftment into at least 80%, more preferably 90%, even more preferably 95% of said patients.

As previously disclosed, the present invention more particularly relates to a batch of T-cells with averaged potency comprising at least one disrupted gene encoding a T cell receptor component, wherein T cells are originated from at least two different donors. A cell according to the present invention refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response. Cell according to the present invention is preferably a T-cell obtained from a donor. Said T cell according to the present invention can be derived from a stem cell. The stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34+ cells. Said isolated cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment, said cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Prior to expansion and genetic modification of the cells of the invention, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art, may be used. In another embodiment, said cell is preferably derived from a healthy donor. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics.

A batch is considered here as the result of pooling different cells from different donor samples.

The cells, as part of the batch, can be cryopreserved in infusible cryomedia, for instance in CryoMACS® freezing bags (Miltenyi Biotec Inc. 120 Presidential Way, Suite 305 Woburn, Mass. 01801). The volume of the bag can vary depending on the intended use (fractionation into multiple doses for different patients or for one patient). Usually each bag contains an aliquot of cryomedia containing the following infusible grade reagents (% vol/vol): 31.25 plasmalyte-A, 31.25 dextrose (5%), 0.45 NaCl, up to 7.50 DMASO, 1.00 dextran 40, 5,00 human serum albumin with approximately between 1.10⁹ and 1.10¹¹ cells, more generally between 1.5 10⁹ and 1.10¹¹, an more specifically 1.5 10⁹ and 1.5. 10¹⁹ cells per bag. Bags, which have in general 10 to 100 ml capacity can be stored in blood bank conditions in a monitored −135° C. freezer or at −196° C. in liquid nitrogen until needed.

Therapeutic Applications

In a preferred embodiment of the present invention, batch of T-cell obtained by the different methods as previously described can be used in allogeneic adoptive cell immunotherapy.

In particular, said batch of T-cell according to the present invention can be used for treating cancer, autoimmune disease or viral infection in a patient in need thereof. In another aspect, the present invention relies on methods for treating patients in need thereof, said method comprising at least one of the following steps:

-   -   (a) providing a batch of T cell with averaged potency obtainable         by any one of the methods previously described;     -   (b) Administrating said cells to said patient.

Advantageously, said T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

Said treatment can be ameliorating, curative or prophylactic. The invention is particularly suited for allogeneic immunotherapy, insofar as it enables the transformation of T-cells, typically obtained from donors, into non-alloreactive cells. This may be done under standard protocols and reproduced as many times as needed. The resulting modified T cells are pooled and administrated to one or several patients, being made available as an “off the shelf” therapeutic product.

Cells that can be used with the disclosed methods are described in the previous section. Said treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorders or Graft versus Host Disease (GvHD). Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the batch of T-cell of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

It can be a treatment in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

According to a preferred embodiment of the invention, said treatment is administrated into patients undergoing an immunosuppressive treatment. The present invention preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent or a chemotherapeutic agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent or chemotherapeutic agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.

The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermaliy, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

The administration of the cells or population of cells can consist of the administration of 10³-10¹⁰ cells per kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In another embodiment, said effective amount of cells are administrated as a single dose. In another embodiment, said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

In another embodiment, said effective amount of cells or pharmaceutical composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p7056 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 11; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

Pharmaceutical Composition

The batch of T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise T-cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration. Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

Definitions

In the description above, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the present embodiments.

As used herein, “a” or “an” may mean one or more than one.

-   -   Amino acid residues in a polypeptide sequence are designated         herein according to the one-letter code, in which, for example,         Q means Gln or Glutamine residue, R means Arg or Arginine         residue and D means Asp or Aspartic acid residue.     -   Nucleotides are designated as follows: one-letter code is used         for designating the base of a nucleoside: a is adenine, t is         thymine, c is cytosine, and g is guanine. For the degenerated         nucleotides, r represents g or a (purine nucleotides), k         represents g or t, s represents g or c, w represents a or t, m         represents a or c, y represents t or c (pyrimidine nucleotides),         d represents g, a or t, v represents g, a or c, b represents g,         t or c, h represents a, t or c, and n represents g, a, t or c.     -   As used herein, “nucleic acid” or “nucleic acid molecule” refers         to nucleotides and/or polynucleotides, such as deoxyribonucleic         acid (DNA) or ribonucleic acid (RNA), oligonucleotides,         fragments generated by the polymerase chain reaction (PCR), and         fragments generated by any of ligation, scission, endonuclease         action, and exonuclease action. Nucleic acid molecules can be         composed of monomers that are naturally-occurring nucleotides         (such as DNA and RNA), or analogs of naturally-occurring         nucleotides (e.g., enantiomeric forms of naturally-occurring         nucleotides), or a combination of both. Nucleic acids can be         either single stranded or double stranded.     -   By “gene” is meant the basic unit of heredity, consisting of a         segment of DNA arranged in a linear manner along a chromosome,         which codes for a specific protein or segment of protein. A gene         typically includes a promoter, a 5′ untranslated region, one or         more coding sequences (exons), optionally introns, a 3′         untranslated region. The gene may further comprise a terminator,         enhancers and/or silencers.     -   The term “transgene” means a nucleic acid sequence (encoding,         e.g., one or more polypeptides), which is partly or entirely         heterologous, i.e., foreign, to the host cell into which it is         introduced, or, is homologous to an endogenous gene of the host         cell into which it is introduced, but which can be designed to         be inserted, or can be inserted, into the cell genome in such a         way as to alter the genome of the cell into which it is inserted         (e.g., it is inserted at a location which differs from that of         the natural gene or its insertion results in a knockout). A         transgene can include one or more transcriptional regulatory         sequences and any other nucleic acid, such as introns, that may         be necessary for optimal expression of the selected nucleic acid         encoding polypeptide. The polypeptide encoded by the transgene         can be either not expressed, or expressed but not biologically         active, in cells in which the transgene is inserted.     -   By “genome” it is meant the entire genetic material contained in         a cell such as nuclear genome, chloroplastic genome,         mitochondrial genome.     -   The term “rare-cutting endonuclease” refers to a wild type or         variant enzyme capable of catalyzing the hydrolysis (cleavage)         of bonds between nucleic acids within a DNA or RNA molecule,         preferably a DNA molecule. Particularly, said nuclease can be an         endonuclease, more preferably a rare-cutting endonuclease which         is highly specific, recognizing nucleic acid target sites         ranging from 10 to 45 base pairs (bp) in length, usually ranging         from 10 to 35 base pairs in length. The endonuclease according         to the present invention recognizes and cleaves nucleic acid at         specific polynucleotide sequences, further referred to as         “target sequence”. The rare-cutting endonuclease can recognize         and generate a single- or double-strand break at specific         polynucleotides sequences.

In a particular embodiment, said rare-cutting endonuclease according to the present invention can be a Cas9 endonuclease. Indeed, recently a new genome engineering tool has been developed based on the RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. CRISPR-mediated genome engineering first proceeds by the selection of target sequence often flanked by a short sequence motif, referred as the proto-spacer adjacent motif (PAM). Following target sequence selection, a specific crRNA, complementary to this target sequence is engineered. Trans-activating crRNA (tracrRNA) required in the CRISPR type II systems paired to the crRNA and bound to the provided Cas9 protein. Cas9 acts as a molecular anchor facilitating the base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. In addition to the target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif—PAM). Following pairing between the dual-RNA and the target sequence, Cas9 subsequently introduces a blunt double strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010). In the present invention, guide RNA can be designed for example to specifically target a gene encoding a TCR component. Following the pairing between the guide RNA and the target sequence, Cas9 induce a cleavage within TCR gene.

Rare-cutting endonuclease can also be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease. Preferred homing endonuclease according to the present invention can be an I-Crel variant. A “variant” endonuclease, i.e. an endonuclease that does not naturally exist in nature and that is obtained by genetic engineering or by random mutagenesis can bind DNA sequences different from that recognized by wild-type endonucleases (see international application WO2006/097854).

Said rare-cutting endonuclease can be a modular DNA binding nuclease. By modular DNA binding nuclease is meant any fusion proteins comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain or protein specifying a nucleic acid target sequence. The DNA binding domain is generally a RNA or DNA-binding domain formed by an independently folded polypeptide that contains at least one motif that recognizes double- or single-stranded. Many such polypeptides have been described in the art having the ability to bind specific nucleic acid sequences. Such binding domains often comprise, as non limiting examples, helix-turn helix domains, leucine zipper domains, winged helix domains, helix-loop-helix domains, HMG-box domains, Immunoglobin domains, B3 domain or engineered zinc finger domain.

According to a preferred embodiment of the invention, the DNA binding domain is derived from a Transcription Activator like Effector (TALE), wherein sequence specificity is driven by a series of 33-35 amino acids repeats originating from Xanthomonas or Ralstonia bacterial proteins. These repeats differ essentially by two amino acids positions that specify an interaction with a base pair (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair in the DNA target is contacted by a single repeat, with the specificity resulting from the two variant amino acids of the repeat (the so-called repeat variable dipeptide, RVD). TALE binding domains may further comprise an N-terminal translocation domain responsible for the requirement of a first thymine base (T₀) of the targeted sequence and a C-terminal domain that containing a nuclear localization signals (NLS). A TALE nucleic acid binding domain generally corresponds to an engineered core TALE scaffold comprising a plurality of TALE repeat sequences, each repeat comprising a RVD specific to each nucleotides base of a TALE recognition site. In the present invention, each TALE repeat sequence of said core scaffold is made of 30 to 42 amino acids, more preferably 33 or 34 wherein two critical amino acids (the so-called repeat variable dipeptide, RVD) located at positions 12 and 13 mediates the recognition of one nucleotide of said TALE binding site sequence; equivalent two critical amino acids can be located at positions other than 12 and 13 specially in TALE repeat sequence taller than 33 or 34 amino acids long. Preferably, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A. In another embodiment, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. A TALE nucleic acid binding domain usually comprises between 8 and 30 TALE repeat sequences. More preferably, said core scaffold of the present invention comprises between 8 and 20 TALE repeat sequences; again more preferably 15 TALE repeat sequences. It can also comprise an additional single truncated TALE repeat sequence made of 20 amino acids located at the C-terminus of said set of TALE repeat sequences, i.e. an additional C-terminal half-TALE repeat sequence.

Other engineered DNA binding domains are modular base-per-base specific nucleic acid binding domains (MBBBD) (PCT/US2013/051783). Said MBBBD can be engineered, for instance, from the newly identified proteins, namely EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins from the recently sequenced genome of the endosymbiont fungi Burkholderia Rhizoxinica (Lackner, Moebius et al. 2011). MBBBD proteins comprise modules of about 31 to 33 amino acids that are base specific. These modules display less than 40% sequence identity with Xanthomonas TALE common repeats, whereas they present more polypeptides sequence variability. When they are assembled together, these modular polypeptides can although target specific nucleic acid sequences in a quite similar fashion as Xanthomonas TALE-nucleases. According to a preferred embodiment of the present invention, said DNA binding domain is an engineered MBBBD binding domain comprising between 10 and 30 modules, preferably between 16 and 20 modules. The different domains from the above proteins (modules, N and C-terminals) from Burkholderia and Xanthomonas are useful to engineer new proteins or scaffolds having binding properties to specific nucleic acid sequences. In particular, additional N-terminal and C-terminal domains of engineered MBBBD can be derived from natural TALE like AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples.

“TALE-nuclease” or “MBBBD-nuclease” refers to engineered proteins resulting from the fusion of a DNA binding domain typically derived from Transcription Activator like Effector proteins (TALE) or MBBBD binding domain, with an endonuclease catalytic domain. Such catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, like for instance I-Tevl, ColE7, NucA and Fok-I. In a particular embodiment, said nuclease is a monomeric TALE-Nuclease or MBBBD-nuclease. A monomeric Nuclease is a nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered DNA binding domain with the catalytic domain of I-Tevl described in WO2012138927. In another particular embodiment, said rare-cutting endonuclease is a dimeric TALE-nuclease or MBBBD-nuclease, preferably comprising a DNA binding domain fused to Fokl. TALE-nuclease have been already described and used to stimulate gene targeting and gene modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010). Such engineered TALE-nucleases are commercially available under the trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

-   -   The term “cleavage” refers to the breakage of the covalent         backbone of a polynucleotide. Cleavage can be initiated by a         variety of methods including, but not limited to, enzymatic or         chemical hydrolysis of a phosphodiester bond. Both         single-stranded cleavage and double-stranded cleavage are         possible, and double-stranded cleavage can occur as a result of         two distinct single-stranded cleavage events. Double stranded         DNA, RNA, or DNA/RNA hybrid cleavage can result in the         production of either blunt ends or staggered ends.     -   By “chimeric antigen receptor” (CAR) it is meant a chimeric         receptor which comprises an extracellular ligand-binding domain,         a transmembrane domain and a signaling transducing domain.

The term “extracellular ligand-binding domain” as used herein is defined as an oligo- or polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

In a preferred embodiment, said extracellular ligand-binding domain comprises a single chain antibody fragment (scFv) comprising the light (V_(L)) and the heavy (V_(H)) variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker. In a preferred embodiment, said scFV is derived from a CD19 antibody. Preferably, said scFV of the present invention comprises a scFV derived from a CD19 monoclonal antibody 4G7 (Peipp, Saul et al. 2004)

The signal transducing domain or intracellular signaling domain of the CAR according to the present invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. Preferred examples of signal transducing domain for use in a CAR can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement. Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. In particular embodiment the signal transduction domain of the CAR of the present invention comprises a co-stimulatory signal molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.

The CAR according to the present invention is expressed on the surface membrane of the cell. Thus, the CAR can comprise a transmembrane domain. The distinguishing features of appropriate transmembrane domains comprise the ability to be expressed at the surface of a cell, preferably in the present invention an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and to interact together for directing cellular response of immune cell against a predefined target cell. The transmembrane domain can further comprise a stalk region_between said extracellular ligand-binding domain and said transmembrane domain. The term “stalk region” used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain. A stalk region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence.

Downregulation or mutation of target antigens is commonly observed in cancer cells, creating antigen-loss escape variants. Thus, to offset tumor escape and render immune cells more specific to target, the CD19 specific CAR can comprise another extracellular ligand-binding domains, to simultaneously bind different elements in target thereby augmenting immune cell activation and function. In one embodiment, the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker. In another embodiment, said different extracellular ligand-binding domains can be placed on different transmembrane polypeptides composing the CAR. In another embodiment, the present invention relates to a population of CARs comprising each one different extracellular ligand binding domains. In a particular, the present invention relates to a method of engineering immune cells comprising providing an immune cell and expressing at the surface of said cell a population of CAR each one comprising different extracellular ligand binding domains. In another particular embodiment, the present invention relates to a method of engineering an immune cell comprising providing an immune cell and introducing into said cell polynucleotides encoding polypeptides composing a population of CAR each one comprising different extracellular ligand binding domains. By population of CARs, it is meant at least two, three, four, five, six or more CARs each one comprising different extracellular ligand binding domains. The different extracellular ligand binding domains according to the present invention can preferably simultaneously bind different elements in target thereby augmenting immune cell activation and function. The present invention also relates to an isolated immune cell which comprises a population of CARs each one comprising different extracellular ligand binding domains.

-   -   The terms “therapeutic agent”, “chemotherapeutic agent”, or         “drug” as used herein refers to a compound or a derivative         thereof that can interact with a cancer cell, thereby reducing         the proliferative status of the cell and/or killing the cell.         Examples of chemotherapeutic agents include, but are not limited         to, alkylating agents (e.g., cyclophosphamide, ifosamide),         metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil         or derivatives thereof), antitumor antibiotics (e.g., mitomycin,         adriamycin), plant-derived antitumor agents (e.g., vincristine,         vindesine, Taxol), cisplatin, carboplatin, etoposide, and the         like. Such agents may further include, but are not limited to,         the anti-cancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™,         RALTRITREXED™, S-(4-Nitrobenzyl)-6-thioinosine (NBMPR),         6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) and         CAMPTOTHECIN™, or a therapeutic derivative of any thereof.     -   By “drug resistant gene” it is meant a nucleic acid sequence         that encodes “resistance” to an agent, such as a         chemotherapeutic agent (e.g. methotrexate). In other words, the         expression of the drug resistant gene in a cell permits         proliferation of the cells in the presence of the agent to a         greater extent than the proliferation of a corresponding cell         without the drug resistant gene. A drug resistant gene of the         invention can encode resistance to anti-metabolite,         methotrexate, vinblastine, cisplatin, alkylating agents,         anthracyclines, cytotoxic antibiotics, anti-immunophilins, their         analogs or derivatives, and the like.

Several drug resistant genes have been identified that can potentially be used to confer drug resistance to targeted cells, and advances in gene therapy techniques (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al. 2003; Zielske, Reese et al. 2003; Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

One example of drug resistant gene can also be a mutant or modified form of Dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in the cell and is essential to DNA synthesis. Folate analogs such as methotrexate (MTX) inhibit DHFR and are thus used as anti-neoplastic agents in clinic. Different mutant forms of DHFR which have increased resistance to inhibition by anti-folates used in therapy have been described. In a particular embodiment, the drug resistance gene according to the present invention can be a nucleic acid sequence encoding a mutant form of human wild type DHFR (GenBank: AAH71996.1) which comprises at least one mutation conferring resistance to an anti-folate treatment, such as methotrexate. In particular embodiment, mutant form of DHFR comprises at least one mutated amino acid at position G15, L22, F31 or F34, preferably at positions L22 or F31 ((Schweitzer, Dicker et al. 1990); International application WO94/24277; U.S. Pat. No. 6,642,043). In a particular embodiment, said DHFR mutant form comprises two mutated amino acids at position L22 and F31. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type DHFR polypeptide set forth in SEQ ID NO: 42. In a particular embodiment, the serine residue at position 15 is preferably replaced with a tryptophane residue. In another particular embodiment, the leucine residue at position 22 is preferably replaced with an amino acid which will disrupt binding of the mutant DHFR to antifolates, preferably with uncharged amino acid residues such as phenylalanine or tyrosine. In another particular embodiment, the phenylalanine residue at positions 31 or 34 is preferably replaced with a small hydrophilic amino acid such as alanine, serine or glycine.

As used herein, “antifolate agent” or “folate analogs” refers to a molecule directed to interfere with the folate metabolic pathway at some level. Examples of antifolate agents include, e.g., methotrexate (MTX); aminopterin; trimetrexate (Neutrexin™); edatrexate; N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex), 5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid (DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF, lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX (10-propargyl-10-deazaaminopterin).

Another example of drug resistance gene can also be a mutant or modified form of ionisine-5′-monophosphate dehydrogenase II (IMPDH2), a rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or modified form of IMPDH2 is an IMPDH inhibitor resistance gene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at least one, preferably two mutations in the MAP binding site of the wild type human IMPDH2 (SEQ ID NO: 43; NP_000875.2) that lead to a significantly increased resistance to IMPDH inhibitor. The mutations are preferably at positions T333 and/or 5351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, the threonine residue at position 333 is replaced with a isoleucine residue and the serine residue at position 351 is replaced with a tyrosine residue. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human IMPDH2 polypeptide set forth in SEQ ID NO: 43.

Another drug resistance gene is the mutant form of calcineurin. Calcineurin (PP2B) is a ubiquitously expressed serine/threonine protein phosphatase that is involved in many biological processes and which is central to T-cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After engagement of the T-cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and active key target gene such as IL2. FK506 in complex with FKBP12, or CsA in complex with CyPA block NFAT access to calcineurin's active site, preventing its dephosphorylation and thereby inhibiting T-cell activation (Brewin, Mancao et al. 2009). The drug resistant gene of the present invention can be a nucleic acid sequence encoding a mutant form of calcineurin resistant to calcineurin inhibitor such as FK506 and/or CsA. In a particular embodiment, said mutant form can comprise at least one mutated amino acid of the wild type calcineurin heterodimer a at positions: V314, Y341, M347, T351, W352, L354, K360, preferably double mutations at positions T351 and L354 or V314 and Y341. In a particular embodiment, the valine residue at position 341 can be replaced with a lysine or an arginine residue, the tyrosine residue at position 341 can be replaced with a phenylalanine residue; the methionine at position 347 can be replaced with the glutamic acid, arginine or tryptophane residue; the threonine at position 351 can be replaced with the glutamic acid residue; the tryptophane residue at position 352 can be replaced with a cysteine, glutamic acid or alanine residue, the serine at position 353 can be replaced with the histidine or asparagines residue, the leucine at position 354 can be replaced with an alanine residue; the lysine at position 360 can be replaced with an alanine or phenylalanine residue of SEQ ID NO: 44. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer a polypeptide set forth in SEQ ID NO: 44 (Gen Bank: ACX34092.1).

In another particular embodiment, said mutant form can comprise at least one mutated amino acid of the wild type calcineurin heterodimer b at positions: V120, N123, L124 or K125, preferably double mutations at positions L124 and K125. In a particular embodiment, the valine at position 120 can be replaced with a serine, an aspartic acid, phenylalanine or leucine residue; the asparagines at position 123 can be replaced with a tryptophane, lysine, phenylalanine, arginine, histidine or serine; the leucine at position 124 can be replaced with a threonine residue; the lysine at position 125 can be replaced with an alanine, a glutamic acid, tryptophane, or two residues such as leucine-arginine or isoleucine-glutamic acid can be added after the lysine at position 125 in the amino acid sequence SEQ ID NO: 45. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer b polypeptide set forth in SEQ ID NO:45 (Gen Bank: ACX34095.1).

Another drug resistant gene is 0(6)-methylguanine methyltransferase (MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents, such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an inhibitor of AGT that potentiates nitrosourea toxicity and is co-administered with TMZ to potentiate the cytotoxic effects of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to inactivation by 6-BG, but retain their ability to repair DNA damage (Maze, Kurpad et al. 1999). In a particular embodiment, AGT mutant form can comprise a mutated amino acid of the wild type AGT position P140, in the amino acid sequence SEQ ID NO: 46 (UniProtKB: P16455). In a preferred embodiment, said proline at position 140 is replaced with a lysine residue.

Another drug resistant gene can be multidrug resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein, known as P-glycoprotein (P-GP) involved in the transport of metabolic byproducts across the cell membrane. The P-Gp protein displays broad specificity towards several structurally unrelated chemotherapy agents. Thus, drug resistance can be conferred to cells by the expression of nucleic acid sequence that encodes MDR-1 (NP_000918).

-   -   The terms “vector” refer to a nucleic acid molecule capable of         transporting another nucleic acid to which it has been linked. A         “vector” in the present invention includes, but is not limited         to, a viral vector, a plasmid, a RNA vector or a linear or         circular DNA or RNA molecule which may consists of a         chromosomal, non chromosomal, semi-synthetic or synthetic         nucleic acids. Preferred vectors are those capable of autonomous         replication (episomal vector) and/or expression of nucleic acids         to which they are linked (expression vectors). Large numbers of         suitable vectors are known to those of skill in the art and         commercially available.     -   By “delivery vector” is intended any delivery vector which can         be used in the present invention to put into cell contact (i.e         “contacting”) or deliver inside cells or subcellular         compartments (i.e “introducing”) agents/chemicals and molecules         (proteins or nucleic acids) needed in the present invention. It         includes, but is not limited to liposomal delivery vectors,         viral delivery vectors, drug delivery vectors, chemical         carriers, polymeric carriers, lipoplexes, polyplexes,         dendrimers, microbubbles (ultrasound contrast agents),         nanoparticles, emulsions or other appropriate transfer vectors.     -   Viral vectors include retrovirus, adenovirus, parvovirus (e.g.         adenoassociated viruses), coronavirus, negative strand RNA         viruses such as orthomyxovirus (e.g., influenza virus),         rhabdovirus (e.g., rabies and vesicular stomatitis virus),         paramyxovirus (e.g. measles and Sendai), positive strand RNA         viruses such as picornavirus and alphavirus, and double-stranded         DNA viruses including adenovirus, herpesvirus (e.g., Herpes         Simplex virus types 1 and 2, Epstein-Barr virus,         cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and         canarypox). Other viruses include Norwalk virus, togavirus,         flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis         virus, for example. Examples of retroviruses include: avian         leukosis-sarcoma, mammalian C-type, B-type viruses, D type         viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.,         Retroviridae: The viruses and their replication, In Fundamental         Virology, Third Edition, B. N. Fields, et al., Eds.,         Lippincott-Raven Publishers, Philadelphia, 1996).     -   By “lentiviral vector” is meant HIV-Based lentiviral vectors         that are very promising for gene delivery because of their         relatively large packaging capacity, reduced immunogenicity and         their ability to stably transduce with high efficiency a large         range of different cell types. Lentiviral vectors are usually         generated following transient transfection of three (packaging,         envelope and transfer) or more plasmids into producer cells.         Like HIV, lentiviral vectors enter the target cell through the         interaction of viral surface glycoproteins with receptors on the         cell surface. On entry, the viral RNA undergoes reverse         transcription, which is mediated by the viral reverse         transcriptase complex. The product of reverse transcription is a         double-stranded linear viral DNA, which is the substrate for         viral integration in the DNA of infected cells. By “integrative         lentiviral vectors (or LV)”, is meant such vectors as         nonlimiting example, that are able to integrate the genome of a         target cell. At the opposite by “non-integrative lentiviral         vectors (or NILV)” is meant efficient gene delivery vectors that         do not integrate the genome of a target cell through the action         of the virus integrase.     -   By cell or cells is intended any eukaryotic living cells,         primary cells and cell lines derived from these organisms for in         vitro cultures.     -   By “primary cell” or “primary cells” are intended cells taken         directly from living tissue (i.e. biopsy material) and         established for growth in vitro, that have undergone very few         population doublings and are therefore more representative of         the main functional components and characteristics of tissues         from which they are derived from, in comparison to continuous         tumorigenic or artificially immortalized cell lines. As non         limiting examples cell lines can be selected from the group         consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS         cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44         cells; K-562 cells, U-937 cells; MRCS cells; IMR90 cells; Jurkat         cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells;         Hu-h7 cells; Huvec cells; Molt 4 cells.     -   Because some variability may arise from the genomic data from         which these polypeptides derive, and also to take into account         the possibility to substitute some of the amino acids present in         these polypeptides without significant loss of activity         (functional variants), the invention encompasses polypeptides         variants of the above polypeptides that share at least 70%,         preferably at least 80%, more preferably at least 90% and even         more preferably at least 95% identity with the sequences         provided in this patent application.

The present invention is thus drawn to polypeptides comprising a polypeptide sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95% 97% or 99% sequence identity with amino acid sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 15 and SEQ ID NO: 22 to SEQ ID NO: 33 and SEQ ID NO: 40 to 46.

-   -   “identity” refers to sequence identity between two nucleic acid         molecules or polypeptides. Identity can be determined by         comparing a position in each sequence which may be aligned for         purposes of comparison. When a position in the compared sequence         is occupied by the same base, then the molecules are identical         at that position. A degree of similarity or identity between         nucleic acid or amino acid sequences is a function of the number         of identical or matching nucleotides at positions shared by the         nucleic acid sequences. Various alignment algorithms and/or         programs may be used to calculate the identity between two         sequences, including FASTA, or BLAST which are available as a         part of the GCG sequence analysis package (University of         Wisconsin, Madison, Wis.), and can be used with, e.g., default         setting. For example, polypeptides having at least 70%, 85%,         90%, 95%, 98% or 99% identity to specific polypeptides described         herein and preferably exhibiting substantially the same         functions, as well as polynucleotide encoding such polypeptides,         are contemplated.

EXAMPLES

1—Statistical Approach: Reduction of Standard Deviation by Pooling Samples from Different Donors

1.1/Reducing Standard Deviation by Pooling

Given

a

a biological parameter varying in a population of eligible donors. The distribution function of the values of

a

is D, where D(x) is the probability that

a

be equal to x when measuring

a

in a randomly drawn donor. The averaged value (or

mean

) of

a

is E(a), its variance being S(a) and its standard deviation is S(a)^((1/2))

Rather than working from only one individual donor, it is proposed here to work from a (equimolar) mix of several individuals (a

pool

). Assumption is made that there isn't any link between the different values of

a

in these different individuals.

Given

b_(n)

the value of

a

measured for a pool of n individuals drawn from the population of eligible donors. The averaged value of

b_(n)

remains E(a), its variance is S(b_(n))=S(a)×n⁻¹, its standard deviation is S(a)^((1/2))×n^(−(1/2)). The dispersion between the values for

b_(n)

is

improved

by factor n^(−(1/2)) in comparison with that observed with

a

between individual donors.

For example, if the biological parameter varies from its mean value with a standard deviation of 10% of that value, then said biological parameter will vary with a standard deviation by 10%/2=5% among pools of 4 individuals, and of 10%/4=2.5% among pools of 16 individuals.

Note that, when reducing the standard deviation of several biological parameters at the same time, if some of them are not independent from each other and donors are selected in order to reduce standard deviations of some of said biological parameters, then it will be possible to observe shifted mean values for some parameters (their standard deviations still being reduced).

1.2/Limits of Pooling: Inclusion of Ineligible Donors

Given

c

a parameter measured in donors susceptible to have values leading to the exclusion of said donors (

c

being subject to a quantitative or binary measure).

Given H(c) the probability that c has a value that makes the donor eligible and (1-H(c)) the probability that c has a value that makes that donor ineligible.

The probability of drawing a pool of n donors without any ineligible donor due to its value for

c

is: H(c)^(n).

Assuming that T, the total number of donors to be included in pools (for the purpose of the required therapeutic doses), then R=T/n represents the number of different pools drawn from the donor population.

Optimizing n, the pool size, is a compromise between:

-   (i) sufficient reduction of the standard deviations relevant for the     intended use of the cells; -   (ii) sufficient probability of not including donors that would     disqualify one pool.     1.3/Example of Hypotheses Sets     Hypotheses Set 1 -   (i) The biological parameters usually measured in the blood from a     healthy donor, the affinities of biological receptors (polymorphs)     to their ligands and the relative proportions of cell subpopulations     within samples coming from donors, have variations of one order of     magnitude at most. In the worst case, the standard deviation of each     parameter will be Vmax=90% of its mean value; -   (ii) the tolerance to these parameters as part of the process or the     therapy is in the order of Vtol=30% (i.e. a desired reduction by at     least a factor of 3); -   (iii) the probability that a candidate donor has a parameter value     that disqualifies any pool that would include it is at most Z=1%; -   (iv) the goal is to have at least K=90% of chances of not including     any disqualifying donor in each of the pools.

According to the above assumptions, the acceptable values for n (i.e. the pool size) can be ranged as follows: Vmax×n ^(−(1/2))≤Vtol⇔n≥(Vmax/Vtol)²;thus:n≥9 and, on another hand, (1−Z)^(n) ≥K⇔n≤ln(K)/ln(1−Z);thus,n≤10 Hypotheses Set 2

This second example focuses on a number of

important

parameters. It is assumed that a donor will disqualify any pool that would include it if any of the p

important

parameters which standard deviations are to be reduced, has a value for that donor that goes beyond Vconf (a confidence interval) around the mean value of said parameter. In other words, homogeneous

average

donors are desired in a sufficient number to have a reduced standard deviation (also referred to as

center of Gaussian

).

Given a₁, a₂, a₃, . . . a_(p), the p parameters which both are to have their standard deviation reduced and their extremes values excluded; given Q_(Vconf)(a_(i)) the probability that the parameter

a_(i)

value be in the confidence interval Vconf_(i);

then, W=ΠQ_(Vconf)(a_(i)) (the product of all Q_(Vconf)(a_(i))) represents the probability that a donor is not disqualifying for a pool that would include it (under the hypothesis that parameters a_(i) are independent). With the preceding notations, (1−Z)=W.

In addition, W^(n) represents the probability of not including any disqualifying donor into a pool of n donors randomly drawn.

If, for example,

-   -   the distributions of p parameters a₁, a₂, a₃, . . . a_(p) are         Gaussian-shaped, and     -   Vconf_(i)=S(a_(i))^((1/2))=Vconf (the same for all p parameters:         one standard deviation),         then, W=Q_(Vconf)(a₁)^(P), the objective being         W^(n)≥K⇔np≤ln(K)/ln(Q_(Vconf))

If

average

donors are sought with respect to p parameters, then no more donors than ln(K)/(ln(Q_(Vconf))×p) should be pooled at the same time.

For Example, with: Q _(Vconf)(a _(i))=84.13%=Q _(Vconf) for any i, and

-   -   K=50%, then one must have n≤4/p

If Vconf corresponds to 2 standard deviations, then Q_(Vconf)(a_(i))=97.72%=Q_(Vconf) for any i and

-   -   with K=50%, then one must have n≤30/p,     -   with K=85%, then one must have n≤7/p.         Hypotheses Set 3

Assuming that:

-   -   3 dominant parameters may qualify or disqualify donors for         pooling;     -   values within +/−2 standard deviations of the abovementioned 3         parameters are tolerated;     -   the probability of not including any donor that would have any         of the abovementioned 3 parameters beyond +/−2 standard         deviations should be at least 75%.

Under these hypotheses, p=3, Q_(Vconf)(a_(i))=97.72%=Q_(Vconf), and K=75%,

Then, n≤4.15 and thus, with n=4, pooling

improves

standard deviations by only a factor of 4^(−(1/2)), that is, by a coefficient of 0.5.

1.4 Conclusion

In view of the above, it seems possible to either define:

-   (I)—an acceptable and large enough interval W (     Center of Gaussian     ) to allow only a weak fraction of ineligible donors, and     -   a minimal acceptable probability K of not including an         ineligible donor in a pool randomly drawn (and thus not to         reject said pool),     -   then, to deduce         F         , the desired reduction factor from the variability between the         donors down to the tolerable variability for pools; and deducing         therefrom:         -   F², the minimal number of donors in each pool;         -   ln(K)/ln(W), the maximal number of donors in each pool

For example, if:

-   -   the distribution of the parameter is Gaussian-shaped,     -   donors are accepted with a parameter value deviating no more         than two standard deviations from the average (for example: with         W=97.72%, there would be a 2.28% probability to reject a         randomly drawn donor),     -   no more than 15% of the pools should be rejected due to their         including a ineligible donor (i.e. K=85%),         then, the maximal number of random donors by pool should be         n=ln(K)/ln(W)≈7.         or:

-   (II) to reduce variability of the parameter by:     -   selecting donors on the basis of said parameter or a surrogate         thereof (e.g. excluding donors bearing extreme values) reducing         thereby the probability for one donor to be ineligible (for         example that probability could be below 0.2% if the exclusion of         ineligible donors in not perfect; which would make W=99.8%);     -   and then, reduce the parameter variability by pooling (i.e. with         the figures abovementioned, n=ln(K)/ln(W)≈81, the standard         deviation of the parameter between pools becomes 9 times smaller         than it is between selected donors)

For example, if the measurement of the parameter is itself subject to some error rate or uncertainty of magnitude

ε

, then selection of donors with a measured parameter within one standard deviation away from the average could allow some ineligible donors (the measured parameters of which would be at most at a distance

ε

outside the required interval) in the pools.

If this probability is of about 0.1%, and the desired probability of not including ineligible donors in a pool is of 90%, then, n=ln(K)/ln(W)≈105 at most.

In this later case, the probability that the parameter value for a pool that would include one ineligible donor be itself outside the desired interval used to select eligible donors (i.e. one standard deviation away from the average in the example hereabove) is extremely low (if not negligible).

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The invention claimed is:
 1. A method for generating a batch of T-cells originating from different human donors, said method comprising the steps of: (a) obtaining T-cell samples from individual human donors; (b) introducing a rare-cutting endonuclease disrupting at least one gene encoding a T cell Receptor (TCR) component into the cells of each individual sample; (c) purifying the TCR negative cells from the sample; and (d) pooling the TCR negative cells originating from the samples from 3 to 50 individual donors; to obtain a batch of T-cells to be used off-the-shelf.
 2. The method according to claim 1, comprising pooling samples from 3 to 10 individual donors.
 3. The method according to claim 1, comprising pooling samples from 3 to 30 individual donors.
 4. The method according to claim 1, wherein said donors express different HLA types.
 5. The method according to claim 1, wherein said method further comprises a step of activation of the T-cells.
 6. The method according to claim 5, wherein said step of activation comprises putting into contact the T-cells with anti-CD3 and anti-CD28antibodies.
 7. The method according to claim 1, wherein said rare-cutting endonuclease is introduced by transfection of mRNA encoding said rare-cutting endonuclease.
 8. The method according to claim 1, wherein said rare-cutting endonuclease is an engineered TALE-nuclease.
 9. The method according to claim 1, wherein at least one donor has T-cells expressing HLA Class I.
 10. The method of claim 9, wherein at least one donor has T-cells expressing HLA-C1 and HLA-C2 alleles, and Bw4 motif of HLA-A or HLA-B alleles.
 11. The method according to claim 1, further comprising expressing at the surface of the T-cell at least one Chimeric Antigen receptor (CAR).
 12. The method according to claim 1, wherein the T-cells are further modified by inactivating at least one gene encoding an immune-checkpoint gene.
 13. The method according to claim 1, wherein the T-cells are further modified by inactivating at least one gene encoding a target for immunosuppressive agent or chemotherapeutic agent.
 14. The method according to claim 1, wherein said T-cells are transformed with a recombinant suicide gene.
 15. A batch of human T-cells obtainable according to the method of claim
 1. 16. A batch of human T-cells, wherein said T cells comprise at least one disrupted gene encoding a T-Cell Receptor (TCR) component and are originated from 3 to 50 different donors.
 17. The batch of human T-cells according to claim 16, comprising T-cells originating from 3 to 10 different donors.
 18. An adoptive cell immunotherapy method, comprising administering a batch of human T cells according to claim 16 to a patient in need of adoptive cell immunotherapy.
 19. A method of treating cancer, a viral infection or an autoimmune disease, comprising administering a batch of human T cells according to claim 16 to a patient with cancer, a viral infection, or an autoimmune disease.
 20. A pharmaceutical composition comprising a batch of human T-cells according to claim
 16. 